Journal
MOLECULAR PLANT
Volume 7, Issue 1, Pages 156-169Publisher
OXFORD UNIV PRESS
DOI: 10.1093/mp/sst144
Keywords
citrate synthase; mitochondria; cysteine residues; redox regulation; thioredoxin; TCA cycle; Arabidopsis
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Funding
- Lehre@LMU of the Ludwig-Maximilians University Munich
- Deutsche Forschungsgemeinschaft, Germany [FI-1655/1-1]
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Here we have investigated the redox regulation of Arabidopsis citrate synthase. We have identified important cysteine residues affecting total enzyme activity as well as the redox sensitivity of the enzyme. Our results indicate that citrate synthase is redox-regulated by thioredoxin.Citrate synthase has a key role in the tricarboxylic (TCA) cycle of mitochondria of all organisms, as it catalyzes the first committed step which is the fusion of a carboncarbon bond between oxaloacetate and acetyl CoA. The regulation of TCA cycle function is especially important in plants, since mitochondrial activities have to be coordinated with photosynthesis. The posttranslational regulation of TCA cycle activity in plants is thus far almost entirely unexplored. Although several TCA cycle enzymes have been identified as thioredoxin targets in vitro, the existence of any thioredoxin-dependent regulation as known for the Calvin cycle, yet remains to be demonstrated. Here we have investigated the redox regulation of the Arabidopsis citrate synthase enzyme by site-directed mutagenesis of its six cysteine residues. Our results indicate that oxidation inhibits the enzyme activity by the formation of mixed disulfides, as the partially oxidized citrate synthase enzyme forms large redox-dependent aggregates. Furthermore, we were able to demonstrate that thioredoxin can cleave diverse intra- as well as intermolecular disulfide bridges, which strongly enhances the activity of the enzyme. Activity measurements with the cysteine variants of the enzyme revealed important cysteine residues affecting total enzyme activity as well as the redox sensitivity of the enzyme.
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